@article{PatilGentschevAdelfingeretal.2012,
  author    = {Patil, Sandeep S. and Gentschev, Ivaylo and Adelfinger, Marion and Donat, Ulrike and Hess, Michael and Weibel, Stephanie and Nolte, Ingo and Frentzen, Alexa and Szalay, Aladar A.},
  title     = {Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody},
  series = {PLoS One},
  volume    = {7},
  journal   = {PLoS One},
  number    = {10},
  doi       = {10.1371/journal.pone.0047472},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130039},
  pages     = {e47472},
  year      = {2012},
  abstract  = {Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.},
  language  = {en}
}
@article{SchaeferWeibelDonatetal.2012,
  author    = {Sch{\"a}fer, Simon and Weibel, Stephanie and Donat, Ulrike and Zhang, Quian and Aguilar, Richard J. and Chen, Nanhai G. and Szalay, Aladar A.},
  title     = {Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors},
  series = {BMC Cancer},
  volume    = {12},
  journal   = {BMC Cancer},
  number    = {366},
  doi       = {10.1186/1471-2407-12-366},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140800},
  year      = {2012},
  abstract  = {Background Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.},
  language  = {en}
}
@article{SchaeferWeibelDonatetal.2012,
  author    = {Sch{\"a}fer, Simon and Weibel, Stephanie and Donat, Ulrike and Zhang, Qian and Aguilar, Richard J. and Chen, Nanhai G. and Szalay, Aladar A.},
  title     = {Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78220},
  year      = {2012},
  abstract  = {Background: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.},
  subject      = {Biochemie},
  language  = {en}
}
@article{SpiveyDeGiorgiZhaoetal.2012,
  author    = {Spivey, Tara L. and De Giorgi, Valeria and Zhao, Yingdong and Bedognetti, Davide and Pos, Zoltan and Liu, Qiuzhen and Tomei, Sara and Ascierto, Maria Libera and Uccellini, Lorenzo and Reinboth, Jennifer and Chouchane, Lotfi and Stroncek, David F. and Wang, Ena and Marincola, Francesco M.},
  title     = {The stable traits of melanoma genetics: an alternate approach to target discovery},
  series = {BMC Genomics},
  volume    = {13},
  journal   = {BMC Genomics},
  number    = {156},
  doi       = {10.1186/1471-2164-13-156},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131992},
  year      = {2012},
  abstract  = {Background: The weight that gene copy number plays in transcription remains controversial; although in specific cases gene expression correlates with copy number, the relationship cannot be inferred at the global level. We hypothesized that genes steadily expressed by 15 melanoma cell lines (CMs) and their parental tissues (TMs) should be critical for oncogenesis and their expression most frequently influenced by their respective copy number. Results: Functional interpretation of 3,030 transcripts concordantly expressed (Pearson's correlation coefficient p-value < 0.05) by CMs and TMs confirmed an enrichment of functions crucial to oncogenesis. Among them, 968 were expressed according to the transcriptional efficiency predicted by copy number analysis (Pearson's correlation coefficient p-value < 0.05). We named these genes, "genomic delegates" as they represent at the transcriptional level the genetic footprint of individual cancers. We then tested whether the genes could categorize 112 melanoma metastases. Two divergent phenotypes were observed: one with prevalent expression of cancer testis antigens, enhanced cyclin activity, WNT signaling, and a Th17 immune phenotype (Class A). This phenotype expressed, therefore, transcripts previously associated to more aggressive cancer. The second class (B) prevalently expressed genes associated with melanoma signaling including MITF, melanoma differentiation antigens, and displayed a Th1 immune phenotype associated with better prognosis and likelihood to respond to immunotherapy. An intermediate third class (C) was further identified. The three phenotypes were confirmed by unsupervised principal component analysis. Conclusions: This study suggests that clinically relevant phenotypes of melanoma can be retraced to stable oncogenic properties of cancer cells linked to their genetic back bone, and offers a roadmap for uncovering novel targets for tailored anti-cancer therapy.},
  language  = {en}
}
@article{KrehanHeubeckMenzeletal.2012,
  author    = {Krehan, Mario and Heubeck, Christian and Menzel, Nicolas and Seibel, Peter and Sch{\"o}n, Astrid},
  title     = {RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex},
  series = {Nucleic Acids Research},
  volume    = {40},
  journal   = {Nucleic Acids Research},
  number    = {16},
  doi       = {10.1093/nar/gks476},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130648},
  pages     = {7956- 7966},
  year      = {2012},
  abstract  = {RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.},
  language  = {en}
}
@article{GentschevAdelfingerJosupeitetal.2012,
  author    = {Gentschev, Ivaylo and Adelfinger, Marion and Josupeit, Rafael and Rudolph, Stephan and Ehrig, Klaas and Donat, Ulrike and Weibel, Stephanie and Chen, Nanhai G. and Yu, Yong A. and Zhang, Qian and Heisig, Martin and Thamm, Douglas and Stritzker, Jochen and MacNeill, Amy and Szalay, Aladar A.},
  title     = {Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma},
  series = {PLoS One},
  volume    = {7},
  journal   = {PLoS One},
  number    = {5},
  doi       = {10.1371/journal.pone.0037239},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129998},
  year      = {2012},
  abstract  = {Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.},
  language  = {en}
}
@article{WangChenMinevetal.2012,
  author    = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Szalay, Aladar A.},
  title     = {Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells},
  series = {Journal of Translational Medicine},
  volume    = {10},
  journal   = {Journal of Translational Medicine},
  number    = {167},
  doi       = {10.1186/1479-5876-10-167},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130019},
  year      = {2012},
  abstract  = {Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.},
  language  = {en}
}
@article{HaddadChenCarlinetal.2012,
  author    = {Haddad, Dana and Chen, Chun-Hao and Carlin, Sean and Silberhumer, Gerd and Chen, Nanhai G. and Zhang, Qian and Longo, Valerie and Carpenter, Susanne G. and Mittra, Arjun and Carson, Joshua and Au, Joyce and Gonen, Mithat and Zanzonico, Pat B. and Szalay, Aladar A. and Fong, Yuman},
  title     = {Imaging Characteristics, Tissue Distribution, and Spread of a Novel Oncolytic Vaccinia Virus Carrying the Human Sodium Iodide Symporter},
  series = {PLoS One},
  volume    = {7},
  journal   = {PLoS One},
  number    = {8},
  doi       = {10.1371/journal.pone.0041647},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130041},
  pages     = {e41647},
  year      = {2012},
  abstract  = {Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS. Methods: GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide \(^{131}\)I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via \(^{124}\)I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both \(^{124}\)I-PET and 99m-technecium gamma-scintigraphy. Results: GLV-1h153 successfully facilitated time-dependent intracellular uptake of \(^{131}\)I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P < 0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10\(^9\) plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean \% ID/gm of 3.82 \(\pm\) 60.46 (P < 0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV1h153-infected tumors were detected via \(^{124}\)I-PET and 99m-technecium-scintigraphy. Conclusion: GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.},
  language  = {en}
}
@phdthesis{Dill2012,
  author    = {Dill, Holger},
  title     = {Functional characterization of the microRNA-26 family in zebrafish neurogenesis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70757},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Formation oft the central nervous system (CNS) from multipotent neuronal stem cells (NSCs) requires a tightly controlled, step-wise activation of the neuronal gene expression program. Expression of neuronal genes at the transition from neural stem cell to mature neuron (i. e. neuronal cell differentiation) is controlled by the Repressor element 1 (RE1) silencing transcription factor (REST) complex. As a master transcriptional regulator, the REST-complex specifically inhibits expression of neuronal genes in non-neuronal tissues and neuronal progenitor cells. Differentiation of NSCs to mature neurons requires the activation of genes controlled by the REST-complex, but how abrogation of REST-complex mediated repression is achieved during neurogenesis is only poorly understood. MicroRNAs (miRNAs) are a class of small regulatory RNAs that posttranscriptionally control target gene expression. Binding of miRNAs to target sequences in the 3'UTR of mRNAs, leads either to degradation or translational inhibition of the mRNA. Distinct neuronal miRNAs (e.g. miR-124) were shown to modulate REST-complex activity by silencing expression of REST-complex components. Interestingly, these miRNAs are also under transcriptional control of the REST-complex and inactivation of the REST-complex precedes their expression. Hence, additional factors are required for derepression of neuronal genes at the onset of neurogenesis. In this study function of the miR-26 family during neurogenesis of the zebrafish (Danio rerio) was analyzed. Computational target prediction revealed a number of REST-complex components as putative miR-26 targets. One of these predicted target genes, the C-terminal domain small phosphatase 2 (Ctdsp2) was validated as an in vivo target for miR-26b. Ctdsps are important cofactors of REST and suppress neuronal gene expression by dephosphorylating the C-terminal domain (CTD) of RNA polymerase II (Pol II). Interestingly, miR-26b is encoded in an intron of the ctdsp2 primary transcript and is cotranscribed together with its host gene. Hence, miR-26b modulates expression of its host gene ctdsp2 in an intrinsic negative autoregulatory loop. This negative autoregulatory loop is inactive in NSCs because miR-26b biogenesis is inhibited at the precursor level. Generation of mature miR-26b is activated during neurogenesis, where it suppresses Ctdsp2 protein expression and is required for neuronal cell differentiation in vivo. Strikingly, miR-26b is expressed prior to miR-124 during neuronal cell differentiation. Thus, it is reasonable to speculate about a function of miR-26b in early events of neurogenesis. In line with this assumption, knockdown of miR-26b in zebrafish embryos results in downregulation of REST-complex controlled neuronal genes and a block in neuronal cell differentiation, most likely due to aberrant regulation of Ctdsp2 expression. This is evident by reduced numbers of secondary motor neurons compared to control siblings. In contrast, motor neuron progenitor cells and glia cells were not affected by depletion of miR-26b.This study identifies the ctdsp2/miR-26b autoregulatory loop as the first experimentally validated interaction between an intronic miRNA and its host gene transcript. Silencing of ctdsp2 by miR-26b in neurons is possible because biogenesis of the ctdsp2 mRNA and mature mir-26b is uncoupled at the posttranscriptional level. Furthermore the obtained data indicate a cell type specific role for miR-26b in vertebrate neurogenesis and CNS development.},
  subject      = {Zebrab{\"a}rbling},
  language  = {en}
}
@phdthesis{Buckel2012,
  author    = {Buckel, Lisa},
  title     = {Evaluating the combination of oncolytic vaccinia virus and ionizing radiation in therapy of preclinical glioma models},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85309},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30\% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells.},
  subject      = {Gliom},
  language  = {en}
}
@phdthesis{Ehrig2012,
  author    = {Ehrig, Klaas},
  title     = {Effects of stem cell transcription factor-expressing vaccinia viruses in oncolytic virotherapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85139},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Krebserkrankungen bleiben auch im Jahr 2012 die zweith{\"a}uftigste Todesursache in der industralisierten Welt. Zus{\"a}tzlich hat die Etablierung der Krebsstammzell-Hypothese grunds{\"a}tzliche Auswirkungen auf die Erfolgsaussichten konventioneller Krebstherapie, wie Chemotherapie oder Strahlentherapie. Deswegen ist es von gr{\"o}βter Notwendigkeit, dass neue Ans{\"a}tze zur Krebstherapie entwickelt werden, die den Ausgang der Behandlung verbessern und zu weniger Nebenwirkungen f{\"u}hren. Diverse vorklinische Studien haben gezeigt, dass die onkolytische Virotherapie mit Vaccinia-Viren ein potentes und gut tolerierbares neues Werkzeug in der Krebstherapie darstellt. Die Effizienz des Vaccinia-Virus als Therapeutikum allein oder in Kombination mit Strahlen- oder Chemotherapie wird aktuell in mehreren klinischen Studien der Phasen I \& II getestet. Krebsstammzellen und Stammzellen teilen eine Vielzahl von Eigenschaften, wie die F{\"a}higkeit zur Selbst-Erneuerung und Pluripotenz, Stilllegung der Zellproliferation, Resistenz gegen Medikamente oder Bestrahlung, die Expression von diversen Zelloberfl{\"a}chen-molek{\"u}len, die Aktivierung und Hemmung spezifischer Signaltransduktionswege oder die Expression von Stammzell-spezifischen Genen. In dieser Arbeit wurden zwei neue rekombinante Vaccinia-Viren entwickelt, welche die Stammzell-Transkriptionsfaktoren Nanog (GLV-1h205) und Oct4 (GLV-1h208) exprimieren, um tiefere Einblicke in die Rolle dieser Masterregulatoren in der Entstehung von Krebs und ihrem Einfluss auf die onkolytische Virotherapie zu gewinnen. Das Replikationspotential beider Virusst{\"a}mme in menschlichen A549-Zellen und PC-3-Zellen wurde anhand von Replikations-Assays bestimmt. Die Expression der Virus-spezifischen Markergene Ruc-GFP und beta-Galaktosidase, wie auch die Expression der Transkriptionsfaktoren Nanog und Oct4 wurde mit Hilfe von RT-PCR, SDS-PAGE und Western blotting, sowie immunozytochemischen Experimenten nachgewiesen. Des Weiteren wurde der Einfluss einer GLV-1h205-Infektion von A549-Zellen auf den Zellzyklus untersucht. Zudem wurde die Bedeutung der Virus-vermittelten Transkriptionsfaktor-Expression auf die Behandlung von subkutanen A549-Tumoren in einem Xenograft-Modell untersucht. Zur Untersuchung, ob die beobachteten Vorteile in der Behandlung von Lungenadenokarzinomen in M{\"a}usen mit GLV-1h205 Promoter- oder Transkriptionsfaktor-abh{\"a}ngig sind, wurde ein Kontroll-Virus (GLV-1h321) hergestellt, dass f{\"u}r eine unfunktionale Nanog-Mutante codiert. Mittels SDS-PAGE und Western blotting sowie Immunozytochemie wurde die Transgen-Expression analysiert. Ein weitere Aspekt dieser Arbeit war die Fragestellung, ob sich das onkolyische Vaccinia-Virus GLV-1h68 eignet, als neues und weniger invasives Therapeutikum effizient Darmkrebszellen zu infizieren um sich in ihnen zu replizieren und diese anschlieβend zu lysieren. Ein derartiger Therapieansatz w{\"u}rde besonders im Hinblick auf sp{\"a}t diagnostizierten, metastasierenden Darmkrebs eine interessante Behandlungsalternative darstellen. Virale Markergen-expression wurde anhand von Fluoreszenzmikroskopie und FACS-Analyse untersucht. Desweiteren wurde gezeigt, dass die einmalige Administration von GLV-1h68 in mindestens zwei verschiedenen Darmkrebszelllinien zu einer signifikanten Inhibierung des Tumorwachstums in vivo und zu signifikant verbessertem {\"U}berleben f{\"u}hrt. Der Transkriptionsfaktor Klf4 wird zwar stark in ruhenden, ausdifferenzierten Zellen des Darmepithels exprimiert, ist hingegen bei Darmkrebs generell dramatisch herabreguliert. Die Expression von Klf4 f{\"u}hrt zu einem Stop der Zellproliferation und inhibiert die Aktivit{\"a}t des Wnt-Signalweges, indem es im Zellkern an die Transaktivierungsdom{\"a}ne von beta-Catenin bindet. Um die Behandlung von Darmkrebs mit Hilfe onkolytischer Virotherapie weiter zu verbessern, wurden verschiedene Vaccinia-Viren (GLV-1h290-292) erzeugt, die durch verschiedene Promoterst{\"a}rken die Expression unterschiedlicher Mengen an Tumorsuppressor Klf4 vermitteln. Die anf{\"a}ngliche Charakterisierung der drei Virusst{\"a}mme mittels Replikations-Assay, Zytotoxizit{\"a}tstudien, SDS-PAGE und Western blotting, Immunozytochemie sowie die Analyse der Proteinfunktion mit Hilfe von qPCR- und ELISA-Analysen zur Bestimmung von zellul{\"a}rem beta-Catenin, zeigten eine Promoter-abh{\"a}ngige Expression und Wirkung von Klf4. F{\"u}r weitere Analysen wurde das Virus GLV-1h291 gew{\"a}hlt, welches nach Infektion die gr{\"o}βte Menge an Klf4 produziert und zus{\"a}tzlich durch die C-terminale Fusion einer TAT Transduktionsdom{\"a}ne Membran-g{\"a}ngig gemacht (GLV-1h391). Die erhaltenen Befunde machen das Klf4-TAT-kodierende Vaccinia-Virus GLV-1h391 zu einem vielversprechenden Kandidaten f{\"u}r eine Behandlung von Darmkrebs beim Menschen.},
  subject      = {Lungenkrebs},
  language  = {en}
}
@phdthesis{Reinboth2012,
  author    = {Reinboth, Jennifer},
  title     = {Cellular Factors Contributing to Host Cell Permissiveness in Support of Oncolytic Vaccinia Virus Replication},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85392},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {In initial experiments, the well characterized VACV strain GLV-1h68 and three wild-type LIVP isolates were utilized to analyze gene expression in a pair of autologous human melanoma cell lines (888-MEL and 1936 MEL) after infection. Microarray analyses, followed by sequential statistical approaches, characterized human genes whose transcription is affected specifically by VACV infection. In accordance with the literature, those genes were involved in broad cellular functions, such as cell death, protein synthesis and folding, as well as DNA replication, recombination, and repair. In parallel to host gene expression, viral gene expression was evaluated with help of customized VACV array platforms to get better insight over the interplay between VACV and its host. Our main focus was to compare host and viral early events, since virus genome replication occurs early after infection. We observed that viral transcripts segregated in a characteristic time-specific pattern, consistent with the three temporal expression classes of VACV genes, including a group of genes which could be classified as early-stage genes. In this work, comparison of VACV early replication and respective early gene transcription led to the identification of seven viral genes whose expression correlated strictly with replication. We considered the early expression of those seven genes to be representative for VACV replication and we therefore referred to them as viral replication indicators (VRIs). To explore the relationship between host cell transcription and viral replication, we correlated viral (VRI) and human early gene expression. Correlation analysis revealed a subset of 114 human transcripts whose early expression tightly correlated with early VRI expression and thus early viral replication. These 114 human molecules represented an involvement in broad cellular functions. We found at least six out of 114 correlates to be involved in protein ubiquitination or proteasomal function. Another molecule of interest was the serine-threonine protein kinase WNK lysine-deficient protein kinase 1 (WNK1). We discovered that WNK1 features differences on several molecular biological levels associated with permissiveness to VACV infection. In addition to that, a set of human genes was identified with possible predictive value for viral replication in an independent dataset. A further objective of this work was to explore baseline molecular biological variances associated with permissiveness which could help identifying cellular components that contribute to the formation of a permissive phenotype. Therefore, in a subsequent approach, we screened a set of 15 melanoma cell lines (15-MEL) regarding their permissiveness to GLV-1h68, evaluated by GFP expression levels, and classified the top four and lowest four cell lines into high and low permissive group, respectively. Baseline gene transcriptional data, comparing low and highly permissive group, suggest that differences between the two groups are at least in part due to variances in global cellular functions, such as cell cycle, cell growth and proliferation, as well as cell death and survival. We also observed differences in the ubiquitination pathway, which is consistent with our previous results and underlines the importance of this pathway in VACV replication and permissiveness. Moreover, baseline microRNA (miRNA) expression between low and highly permissive group was considered to provide valuable information regarding virus-host co-existence. In our data set, we identified six miRNAs that featured varying baseline expression between low and highly permissive group. Finally, copy number variations (CNVs) between low and highly permissive group were evaluated. In this study, when investigating differences in the chromosomal aberration patterns between low and highly permissive group, we observed frequent segmental amplifications within the low permissive group, whereas the same regions were mostly unchanged in the high group. Taken together, our results highlight a probable correlation between viral replication, early gene expression, and the respective host response and thus a possible involvement of human host factors in viral early replication. Furthermore, we revealed the importance of cellular baseline composition for permissiveness to VACV infection on different molecular biological levels, including mRNA expression, miRNA expression, as well as copy number variations. The characterization of human target genes that influence viral replication could help answering the question of host cell response to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.},
  subject      = {Vaccinia-Virus},
  language  = {en}
}
@article{GrossHennardMasourisetal.2012,
  author    = {Gross, Henrik and Hennard, Christine and Masouris, Ilias and Cassel, Christian and Barth, Stephanie and Stober-Gr{\"a}sser, Ute and Mamiani, Alfredo and Moritz, Bodo and Ostareck, Dirk and Ostareck-Lederer, Antje and Neuenkirchen, Nils and Fischer, Utz and Deng, Wen and Leonhardt, Heinrich and Noessner, Elfriede and Kremmer, Elisabeth and Gr{\"a}sser, Friedrich A.},
  title     = {Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression},
  series = {PLoS One},
  volume    = {7},
  journal   = {PLoS One},
  number    = {8},
  doi       = {10.1371/journal.pone.0042106},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133707},
  year      = {2012},
  abstract  = {The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA-antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV-infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.},
  language  = {en}
}
@article{TomeiAdamsUccellinietal.2012,
  author    = {Tomei, Sara and Adams, Sharon and Uccellini, Lorenzo and Bedognetti, Davide and De Giorgi, Valeria and Erdenebileg, Narnygerel and Libera Ascierto, Maria and Reinboth, Jennifer and Liu, Qiuzhen and Bevilacqua, Generoso and Wang, Ena and Mazzanti, Chiara and Marincola, Francesco M.},
  title     = {Association between HRAS rs12628 and rs112587690 polymorphisms with the risk of melanoma in the North American population},
  series = {Medical Oncology},
  volume    = {29},
  journal   = {Medical Oncology},
  number    = {5},
  doi       = {dx.doi.org/10.1007/s12032-012-0255-3},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126834},
  pages     = {3456-3461},
  year      = {2012},
  abstract  = {HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT- located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case-control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690.},
  language  = {en}
}
@phdthesis{Neuenkirchen2012,
  author    = {Neuenkirchen, Nils},
  title     = {An in vitro system for the biogenesis of small nuclear ribonucleoprotein particles},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Most protein-encoding genes in Eukaryotes are separated into alternating coding and non-coding sequences (exons and introns). Following the transcription of the DNA into pre-messenger RNA (pre-mRNA) in the nucleus, a macromolecular complex termed spliceosome removes the introns and joins the exons to generate mature mRNA that is exported to the cytoplasm. There, it can be interpreted by ribosomes to generate proteins. The spliceosome consists of five small nuclear ribonucleic acids (snRNAs) and more than 150 proteins. Integral components of this complex are RNA-protein particles (RNPs) composed of one or two snRNAs, seven common (Sm) and a various number of snRNP-specific proteins. The Sm proteins form a ring-structure around a conserved site of the snRNA called Sm site. In vitro, Sm proteins (B/B', D1, D2, D3, E, F, G) and snRNA readily assemble to form snRNPs. In the context of the cell, however, two macromolecular trans-acting factors, the PRMT5 (protein arginine methyltransferases type 5) and the SMN (survival motor neuron) complex, are needed to enable this process. Initially, the Sm proteins in the form of heterooligomers D1/D2, D3/B and F/E/G are sequestered by the type II methyltransferase PRMT5. pICln, a component of the PRMT5 complex, readily interacts with Sm proteins to form two distinct complexes. Whereas the first one comprises pICln and D3/B the second one forms a ring consisting of pICln, D1/D2 and F/E/G (6S). It has been found that pICln prevents the premature interaction of snRNAs with the Sm proteins in these complexes and thus functions as an assembly chaperone imposing a kinetic trap upon the further assembly of snRNPs. PRMT5 catalyzes the symmetrical dimethylation of arginine residues in B/B', D1 and D3 increasing their affinity towards the SMN complex. Finally, the SMN complex interacts with the pICln-Sm protein complexes, expels pICln and mediates snRNP assembly in an ATP-dependent reaction. So far, only little is known about the action of PRMT5 in the early phase of snRNP assembly and especially how the 6S complex is formed. Studies of this have so far been hampered by the unavailability of soluble and biologically active PRMT5 enzyme. The composition of the SMN complex and possible functions of individual subunits have been elucidated or hypothesized in recent years. Still, the exact mechanism of the entire machinery forming snRNPs is poorly understood. In vivo, reduced production of functional SMN protein results in the neurodegenerative disease spinal muscular atrophy (SMA). How specific SMN mutations that have been found in SMA patients cause the disease remains elusive, yet, are likely to interfere with either SMN complex stability or snRNP assembly. The aim of this work was to establish an in vitro system to recapitulate the cytoplasmic assembly of snRNPs. This was enabled by the recombinant production of all PRMT5 and SMN complex components as well as Sm proteins in a combination of bacterial and insect cell expression systems. Co-expression of human PRMT5 and its direct interaction partner WD45 (WD-repeat domain 45) in Sf21 (Spodoptera frugiperda 21) insect cells resulted for the first time in soluble and biologically active enzyme. Recombinant PRMT5/WD45 formed complexes with Sm protein heterooligomers as well as pICln-Sm protein complexes but not with F/E/G alone. Also, the enzyme exhibited a type II methyltransferase activity catalyzing the mono- (MMA) and symmetrical dimethylation (sDMA) of Sm proteins B, D1 and D3. Two experimental setups were devised to quantitatively analyze the overall methylation of substrates as well as to identify the type and relative abundance of specific methylation types. Methylation of Sm proteins followed Michaelis-Menten kinetics. Complex reconstitutions and competition of the methylation reaction indicate that 6S is formed in a step-wise manner on the PRMT5 complex. The analysis of the methylation type could be applied to deduce a model of sequential MMA and sDMA formation. It was found that large Sm protein substrate concentrations favored monomethylation. Following a distributive mechanism this leads to the conclusion that PRMT5 most likely confers partial methylation of several different substrate proteins instead of processing a single substrate iteratively until it is completely dimethylated. Finally, the human SMN complex was reconstituted from recombinant sources and was shown to be active in snRNP formation. The introduction of a modified SMN protein carrying a mutation (E134K) present in spinal muscular atrophy (SMA) proved that mutated complexes can be generated in vitro and that these might be applied to elucidate the molecular etiology of this devastating disease.},
  subject      = {Biogenese},
  language  = {en}
}